Nucleic acid amplification reaction station for disposable test devices

ABSTRACT

An instrument for conducting nucleic acid amplification reactions in a disposable test device. The test device includes a first reaction chamber containing a first nucleic acid amplification reagent (e.g., primers and nucleotides) and a second reaction chamber either containing, or in fluid communication, with a second nucleic acid amplification reagent (e.g., an amplification enzyme such as RT). The instrument includes a support structure receiving the test device. A temperature control system maintains the first reaction chamber at a first elevated temperature but simultaneously maintains the second nucleic acid amplification reagent at a second temperature lower than the first temperature so as to preserve the second nucleic acid amplification reagent. An actuator operates on a fluid conduit in the test device to place the first and second reaction chambers in fluid communication with each other after a reaction has occurred in the first reaction chamber at the first temperature. A pneumatic system is also provided that assists in fluid transfer of a reaction solution from the first chamber to the second chamber.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation application of prior application Ser. No.11/180,806 filed Jul. 12, 2005 now U.S. Pat. No. 7,214,529, allowed,which is a continuation of Ser. No. 10/141,049 filed May 7, 2002, nowU.S. Pat. No. 6,949,376, which is a divisional of application Ser. No.09/420,140 filed Oct. 18, 1999 now U.S. Pat. No. 6,429,007, which is acontinuation-in-part of application Ser. No. 09/053,823 filed Apr. 2,1998, now U.S. Pat. No. 5,989,499, which is a continuation-in-part ofapplication Ser. No. 08/850,207 filed May 2, 1997, now U.S. Pat. No.5,786,182. The entire content of the related applications and patentsare fully incorporated by reference herein.

BACKGROUND OF THE INVENTION

A. Field of the Invention

This invention relates to the field of methods and devices forperforming nucleic acid amplification reactions. More particularly, theinvention relates to an automated instrument for performing nucleic acidamplification reactions.

B. Description of Related Art

Nucleic acid based amplification reactions are now widely used inresearch and clinical laboratories for the detection of genetic andinfectious diseases. The currently known amplification schemes can bebroadly grouped into two classes, based on whether, after an initialdenaturing step (typically performed at a temperature of ≧65 degrees C.)for DNA amplifications or for RNA amplifications involving a high amountof initial secondary stricture, the reactions are driven via acontinuous cycling of the temperature between the denaturationtemperature and a primer annealing and amplicon synthesis (or polymeraseactivity) temperature (“cycling reactions”), or whether the temperatureis kept constant throughout the enzymatic amplification process(“isothermal reactions”). Typical cycling reactions are the Polymeraseand Ligase Chain Reaction (PCR and LCR, respectively). Representativeisothermal reaction schemes are NASBA (Nucleic Acid Sequence BasedAmplification). Transcription Mediated Amplification (TMA), and StrandDisplacement Amplification (SDA). In the isothermal reactions, after theinitial denaturation step (if required), the reaction occurs at aconstant temperature, typically a lower temperature at which theenzymatic amplification reaction is optimized.

Prior to the discovery of thermostable enzymes, methodologies that usedtemperature cycling were seriously hampered by the need for dispensingfresh polymerase into an amplification tube (such as a test tube) aftereach denaturation cycle, since the elevated temperature required fordenaturation inactivated the polymerase during each cycle. Aconsiderable simplification of the PCR assay procedure was achieved withthe discovery of the thermostable Taq polymerase (from Thermophilusaquaticus). This improvement eliminated the need to open amplificationtubes after each amplification cycle to add fresh enzyme. This led tothe reduction of both the contamination risk and the enzyme-relatedcosts. The introduction of thermostable enzymes has also allowed therelatively simple automation of the PCR technique. Furthermore, this newenzyme allowed for the implementation of simple disposable devices (suchas a single tube) for use with temperature cycling equipment.

TMA requires the combined activities of at least two (2) enzymes forwhich no optimal thermostable variants have been described. For optimalprimer annealing in the TMA reaction, an initial denaturation step (at atemperature of ≧65 degrees C.) is performed to remove secondarystructure of the target. The reaction mix is then cooled down to atemperature of 42 degrees C. to allow primer annealing. This temperatureis also the optimal reaction temperature for the combined activities ofT7 RNA polymerase and Reverse Transcriptase (RT), which includes anendogenous RNase H activity or is alternatively provided by anotherreagent. The temperature is kept at 42 degrees C. throughout thefollowing isothermal amplification reaction. The denaturation step,which precedes the amplification cycle, however forces the user to addthe enzyme to the test tube after the cool down period in order to avoidinactivation of the enzymes. Therefore, the denaturation step needs tobe performed separately from the amplification step.

In accordance with present practice, after adding the test or controlsample or both to the amplification reagent mix (typically containingthe nucleotides and the primers), the test tube is subject totemperatures≧65 degrees C. and then cooled down to the amplificationtemperature of 42 degrees C. The enzyme is then added manually to startthe amplification reaction. This step typically requires the opening ofthe amplification tube. The opening of the amplification tube to add theenzyme or the subsequent addition of an enzyme to an open tube is notonly inconvenient, it also increases the contamination risk.

An alternative approach to amplification of a DNA sample is described inCorbett et al., U.S. Pat. No. 5,270,183. In this technique, a reactionmixture is injected into a stream of carrier fluid. The carrier fluidthen passes through a plurality of temperature zones in which thepolymerase chain reactions take place. The temperature of the differentzones and the time elapsed aked for the carrier fluid to traverse thetemperature zones is controlled such that three events occur:denaturation of the DNA strands, annealing of oligonucleotine primers tocomplemetary sequences in the DNA, and synthesis of the new DNA strands.A tube and associated temperature zones and pump means are provided tocarry out the '183 patent process.

The present invention provides a nucleic amplification reaction systemthat substantially eliminates the risk of contamination, and provide aconvenient, simple and easy to use approach for nucleic acidamplification reactions. The test devices and amplification station inaccordance with the present invention achieves the integration of thedenaturation step with the amplification step without the need for amanual enzyme transfer and without exposing the amplification chamber tothe environment. The contamination risks from sample to samplecontamination within the processing station are avoided since theamplification reaction chamber is sealed and not opened to introduce thepatient sample to the enzyme. Contamination from environmental sourcesis avoided since the amplification reaction chamber remains sealed. Therisk of contamination in nucleic acid amplification reactions isespecially critical since large amounts of the amplification product areproduced.

SUMMARY OF THE INVENTION

In a first aspect, a station is provided for conducting a nucleic acidamplification reaction that is conducted in a unitary, disposable testdevice. The test device has a first reaction chamber containing a firstnucleic acid amplification reagent (such as primers and nucleotides) anda second reaction chamber either containing, or in fluid communicationwith, a second nucleic acid amplification reagent (e.g., anamplification enzyme such as RT).

The station includes a support structure receiving the test device. Inthe illustrated embodiment, the support structure comprises a set ofraised ridges that receive a disposable test strip containing thereaction chambers. The station further includes a temperature controlsystem for the test device. The temperature control system maintains thefirst reaction chamber at a first elevated temperature, wherein areaction takes place in the first reaction chamber between a fluidsample or target and the first amplification reagent. However, thetemperature control system simultaneously maintains the second nucleicacid amplification reagent at a second temperature lower than said firsttemperature so as to preserve said second nucleic acid amplificationreagent. In the illustrated embodiment, the temperature control systemcomprises a pair of thermo-electric elements coupled to the supportstructure.

The station further comprises an actuator operative on the test deviceto place the first and second reaction chambers in fluid communicationwith each other. The first and second reaction chambers are normallyisolated from each other by a closed valve in a connecting conduitlinking the first and second chambers together. The actuator isoperative on the test device after a reaction has occurred in the firstreaction chamber at the first temperature. A second portion of nucleicaced amplification reaction e.g., amplification of target RNA or DNAsequences in the sample, occurs in the second chamber with the secondnucleic acid amplification reagent. The second nucleic acidamplification reagent is preserved by virtue of maintaining the reagentat the second (i.e. lower) temperature while the reaction in the firstchamber is conducted at the first (e.g. higher) temperature.

As described herein, the amplification station may be designed toprocess a multitude of test devices simultaneously. In this embodiment,the support structure, temperature control system and actuators aredesigned to operate on all of the test devices simultaneously.

After the reaction between the fluid sample and the reagents in thefirst reaction chamber, the reaction solution is directed into thesecond reaction chamber. Several possible mechanisms are contemplatedfor promoting the transfer of the reaction solution to the secondreaction chamber. In one embodiment, vacuum is drawn on the secondreaction chamber in the manner described in our prior U.S. Pat. No.5,786,182. In a more preferred embodiment, the support structure workswith a vacuum housing that is lowered onto the support structure to forma vacuum enclosure around the test devices. A vacuum is drawn in thevacuum enclosure. When the vacuum is released, a pressure gradientbetween the first and second reaction chambers causes the reactionsolution to flow between the first and second reaction chambers.

Thus, in a second aspect of the invention, an amplification station isprovided for conducting a plurality of nucleic acid amplificationreactions in a plurality of disposable test devices. The amplificationstation comprises a support structure adapted to receive a plurality ofsaid test devices and a temperature control system for the test devices,an actuator assembly and a pneumatic system. The temperature controlsystem maintains the temperature of the test devices according to adesired profile (or profiles) for the nucleic acid amplificationreaction. The actuator assembly operates on each of the test devices toopen a fluid conduit in the test devices and thereby allow a reactionsolution to flow from a first location in the test device (e.g., a firstreaction chamber) to a second location in said test device (e.g., asecond reaction chamber containing an amplification enzyme). Thepneumatic system operates on the test devices to draw a reactionsolution from the first location to the second location after theactuator assembly has operated on the test devices to place the firstand second portions in fluid communication with each other.

In one possible embodiment of the invention, the amplification stationincludes a mechanical agitation system agitating the test devices tothereby promote mixing of the reaction solution and the reagents in thefirst and second reaction chambers.

The form factor of the test device processed in the amplificationstation is not considered critical. In the illustrated embodiment thetest device takes the form of a test strip that is compatible with acurrently available analytical fluid transferred detection instrument,namely the VIDAS® (instrument manufactured and distributed by theassignee of the present invention, bioMerieux, Inc. Thus, providing testdevices in a size and form factor to be readily used in an existing orselected instrument base allows the test devices to be widelycommercialized and used with a reduced capital expenditure, and withouthaving to develop a new instrument for processing the reaction anddetecting the resulting amplicons. It will apparent, however, from thefollowing detailed description that the invention can be practiced inother configurations and form factors from the presently preferredembodiment described in detail herein.

These and many other aspects and features of the invention will bereadily understood from the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

A presently preferred embodiment of the invention is described below inconjunction with the appended drawing figures, wherein like referencenumerals refer to like elements in the various views, and in which:

FIG. 1 is a perspective view of an amplification reaction station inaccordance with a preferred embodiment of the invention;

FIG. 1A is a perspective view of a test strip and associated covermember that is used with the inventive amplification reaction station ofFIG. 1;

FIG. 2 is another perspective view of the test strip and cover member ofFIG. 1A, showing the cover member attached to the test strip and with aportion of the cover member in a raised or elevated position, allowingaccess to the first reaction chamber of the dual chamber reaction vesseltherein;

FIG. 3 is another perspective view of the test strip of FIG. 2;

FIG. 4 is an isolated, perspective view of the cover member of FIGS. 2-3shown from below;

FIG. 4A is a perspective view of an alternative embodiment of the covermember of FIG. 4, showing a manually-actuable button is provided topierce the film membrane covering chamber A of the test strip of Figure;

FIG. 4B is an isolated, perspective view of the cover member of FIG. 4Ashown from below, showing a projecting point that pierces the membranewhen the button of FIG. 4A is depressed;

FIG. 5 is a top plan view of the test strip of FIGS. 2-3;

FIG. 6 is a cross-sectional view of the test strip of FIG. 5, shownalong the lines 6-6 of FIG. 5;

FIG. 7 is a cross-sectional vie; of the test strip of FIG. 5, shownalong the lines 7-7 of FIG. 5;

FIG. 8 is a side elevational view of the test strip of FIG. 5;

FIG. 9 is a detailed elevational view of the upper portion of the teststrip in the region adjacent to the second reaction vessel, showing thefeatures on the side of the test strip that are securely gripped by theresilient legs of the cover member to lock the cover member to the teststrip;

FIG. 10 is a detailed cross-sectional view of test strip, partiallybroken away, illustrating the locking features shown in FIG. 9;

FIG. 11 is a detailed plan view of the top of the test strip of FIG. 5in the region of the connecting conduit linking the first reactionchamber to the second reaction chamber;

FIG. 12 is a cross-sectional view of a portion of the test strip ofFIGS. 5 and 11, taken along the lines 12-12 of FIG. 13, that is, alongthe long axis of the test strip in the region of the connecting conduitlinking the first reaction chamber to the second reaction chamber,showing the placement of a ball inside the connecting conduit that actsas a valve to close off the connecting conduit;

FIG. 13 is a cross-sectional view of a portion of the test strip of FIG.5 taken in a direction orthogonal to the long axis of the capsule, alongthe lines 13-13 of FIGS. 11 and 12;

FIG. 14 is a perspective view of a test strip or the kind shown in FIG.5 with a fork implement used to open up the connecting conduit with thearrow indicating the relative motion of the fork with respect to thetest strip and the dotted lines indicating the insertion of the prongsof the fork into the test strip to open the ball valve in the connectingconduit;

FIG. 15 is a schematic illustration of a vacuum station incorporatingheat sinks for the test strip and having a housing that engages asupport structure to form a vacuum enclosure around the test strips,with each test strip associated with a fork for opening the connectingconduit when the vacuum chamber housing moves down and engages thesupport structure;

FIG. 16 is a cross-sectional view of the test strip of FIG. 5 taken in adirection transverse to the long axis of the test strip in the vicinityof the connecting conduit, showing the action of the forks of FIGS. 14and 15 in deforming the material of the connecting conduit to therebyopen the valve;

FIG. 17 is a cross-sectional view of the test strip of FIG. 16, showingthe deformation of the connecting conduit and the flow of fluid throughthe connecting conduit;

FIG. 18 is a perspective view of the instrument of FIG. 1 with the topand side panels removed in order show the details of the two bays andthe pneumatic system;

FIG. 19 is a perspective view of the instrument of FIGS. 1 and 18 asseen from the rear;

FIG. 20 is a perspective view of one of the stations in the instrumentof FIG. 1A, shown isolated from the rest of the instrument in order tobetter illustrate the mechanical features thereof;

FIG. 21 is an elevational view of the station of FIG. 20 shown from therear side thereof;

FIG. 22 is a side elevational view of the station of FIG. 20;

FIG. 23 is another perspective view of the station of FIG. 20;

FIG. 24 is another perspective view of the station of FIG. 20 shown frombelow and to the front of the station, showing the belt drive mechanismsthat control the raising and lowering of the vacuum enclosure housingand the mechanical agitation of the test strips;

FIG. 25 is a side elevational view of the station of FIG. 20, shown fromthe opposite side of FIG. 22;

FIG. 26 is a front elevational view of the station of FIG. 20;

FIG. 27 is a top plan view of the station of FIG. 20;

FIG. 28 is a vertical cross-section of the station of FIG. 20, takenalong the lines 28-28 of FIGS. 25 and 27;

FIGS. 29A and 29B are perspective views of the vacuum housing of FIGS.10-28 that lowers onto the support structure carrying the test strips inorder to form a vacuum enclosure around the test strips;

FIG. 29C is a cross-sectional view of the vacuum housing of FIG. 29A;

FIG. 30A-30D are several views of the actuator assembly of FIG. 28 thatoperates on the valves in the test strips to allow a reaction solutionto flow from the first chamber of the dual chamber reaction vesseldisposed therein to the second chamber;

FIG. 31 is a side view, partially in section, of a portion of the vacuumhousing of FIGS. 29A and 29B in a raised position relative to thesupport structure that holds the test strips;

FIGS. 32A-D are several views of an optical sensor arrangement that ispositioned above the support structure for the purpose of detectingwhether the user has installed a test strip in each of the slots of thesupport structure;

FIGS. 33A, 33B and 33C are several views of the support structure ofFIG. 20 which holds the test strips in the station;

FIG. 34 is a bottom plan view of the support structure of FIG. 33A,showing the position of thermo-electric elements and heat sinks for thetest strips that maintain the dual chamber reaction vessel at the propertemperatures;

FIG. 35 is a schematic illustration of the operation of thethermo-electric elements of FIG. 34;

FIG. 36 is a cross-section of the tray support member of FIG. 33A takenalong the lines 36-36;

FIG. 37 is a cross-section of the tray support member of FIG. 33A takenalong the lines 37-37 of FIG. 34, showing the thermo-electric elementsand the heat sinks;

FIG. 38 is a more detailed cross-sectional view of the support structureof right-hand hand side of FIG. 37;

FIG. 39 is another cross-sectional views of the support structure ofFIG. 33C. taken along the lines 39-39 of FIGS. 33C and 34;

FIG. 40 is a perspective view of the superstructure of the station withmost of the parts thereof removed in order to better illustrate thedrive systems of the station;

FIG. 41A is an isolated perspective view of the horizontal supportmember and lead screw collar of FIG. 28; FIG. 41B is a cross-sectionalview of the support member and collar of FIG. 41A;

FIG. 42 is a perspective view of the drive systems of FIG. 40, shownfrom below;

FIG. 43 is a bottom plan view of the drive systems shown in FIGS. 40 and42;

FIG. 44 is a cross-section of the drive system of FIG. 40, taken alongthe lines 44-44;

FIG. 45 is a schematic diagram of the electrical system for the stationof FIG. 20;

FIG. 46 is a schematic diagram of the pneumatic system for the stationof FIG. 20; and

FIG. 47 is a diagram and chart showing a representative thermal cyclingof the station of FIG. 20.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

I. General Overview

Referring now to FIG. 1, a preferred embodiment of an instrument forcontrolling a nucleic acid amplification reactions in a disposable testdevice is indicated generally by reference numeral 1. A presentlypreferred embodiment of the disposable test device is shown in FIGS.1A-17 and is described at length herein. One or more disposable testdevices, such as one to six or six to twelve of such devices of FIG. 2,are inserted manually into the instrument 1 and installed on supportstructures therein. The disposable test devices contain amplificationreaction chambers, reagents and a sample for a nucleic acidamplification reaction.

The instrument 1 includes an amplification module 2 having two bays 3,designated bay A and bay B. Additional modules containing additionalbays may be added as desired to increase sample throughput. Each bay 3acts as an opening for an amplification station 200 located within theamplification module 2. The amplification stations 200 are shown in moredetail in FIG. 20 et seq. The amplification module 2 includesmechanical, pneumatic, temperature and electrical systems that control anucleic acid amplification reaction occurring in the disposable testdevice of FIG. 2. These systems will be described at length below.

The amplification module 2 is linked via an RS-232 cable 4 to ageneral-purpose computer system 5, which includes a central processingunit 6 and a user interface 7. The CPU 6 is loaded with a softwareprogram that allows a technician to control the operation of the station1 via the user interface 7. In a preferred embodiment the CPU isincorporated in the module 2. More than one amplification module 2 canbe linked to the computer system 5 in a further high capacityimplementation. An additional amplification module having three bays(resulting in a total of five bays) could be linked to the computersystem. The menu screens on the user interface 7 allow the operator tocontrol the operation of each bay in the module 2 or in any extensionmodule that may be added. The described system is versatile and may beadapted to user requirements for various testing situations.

After the nucleic acid amplification reaction has been performed in thedisposable test devices inserted into the bays 3 of the instrument 1,the devices are manually removed from the instrument 1 and transferredto another instrument for hybridizing the amplification products to oneor more probes, for example a detector probe and a capture probe anddetecting the presence of the detector probe with optical techniques. Asuitable instrument for processing the test strips of FIG. 1A is theVIDAS® instrument of bioMerieux Inc.

It will be appreciated that the choice of subsequent analytic instrumentfor processing the test device will depend on the design and form factorof the test device. The present inventive principles of theamplification station are applicable to other form factors, and thus theinvention is not limited to any particular type of test device oranalytic instrument.

The detailed description of the design of the amplification stations 200in the instrument 1 of FIG. 1 will be more readily understood if thereader is already familiar with the design of the test device used bysuch stations, and the theory of operation thereof. Therefore, the nextsection of this document sets forth a detailed description of thedisposable test device of FIG. 1A that is processed by the instrument 1.The operational features of the instrument 1 are fully set forth insubsequent sections of this document, and in the drawings beginning withFIG. 18. Further, it should be noted that both of the amplificationstations 200 located behind the two bays 3 of FIG. 1A are identical, andtherefore this document will only describe one of the amplificationstations. To the extent that the two amplification stations share commoncomponents of a pneumatic or electrical system, those features will alsobe explained.

II. Detailed Discussion of Disposable Test Device Construction andOperation

Referring to FIGS. 1, 1A, and 2-3, the amplification station 200 of FIG.1 is designed to receive a test strip 10 having a dual chamber reactionvessel 12. The reaction vessel 12 has a single or unit dose of reagentsfor a reaction typically requiring differential heat and containmentfeatures, such as a nucleic acid amplification reaction (for example,TMA reaction), packaged ready for use. The dual chamber reaction vesselis designed as a single use disposable unit. The reaction vessel ispreferably integrally molded into a test device, such as a strip 10,having a set of wash reagent and detection wells 13 for use in aseparate amplification reaction (hybridization) product detectionstation. Alternatively, the reaction vessel 12 can be made as a standalone unit with flange or other suitable structures for being able to beinstalled in a designated space provided in such a test device.

In the dual chamber reaction vessel 12, two separate reaction chambers,A and B, are provided. The two main reagents in the vessel for thereaction are stored in a spatially separated fashion. One chamber,chamber A, has the heat stable sample/amplification reagent (containingprimers, nucleotides, and other necessary salts and buffer components),and the other chamber, chamber B, contains the heat labile enzymaticreagents. e.g., T7 and RT. Alternatively, the heat labile enigmaticreagents may be stored in an intermediate chamber or well in fluidcommunication with the second chamber, such that a reaction solutionfrom the first chamber flows through the intermediate chamber en routeto the second chamber.

The two chambers are linked to each other by a fluid channel orconnecting conduit 50 extending from the first chamber to the secondchamber. A means is provided for controlling or allowing the flow offluid through the fluid channel from the first chamber to the secondchamber. Various fluid flow control means are contemplated, such asproviding a valve in the fluid channel, as described in the priorapplication Ser. No. 09/053,823 filed Apr. 2, 1998, now U.S. Pat. No.5,989,499 and U.S. Pat. No. 5,786,182. Several different valveembodiments are described therein.

A technician loads a fluid sample into the first chamber A and installsthe test strip 10 into a bay 3 of the instrument 1 of FIG. 1. Inside theamplification station 200, a thermoelectric temperature control systemheats the first chamber only to a denaturation temperature (e.g., 95degrees C.). After the amplification reagents in the first chamber havereacted with the fluid sample and the denaturation process has beencompleted, the first chamber is quickly cooled to 42 degrees C. forprimer annealing. The two chambers of the reaction vessel are not influid communication with each other prior to completion of thedenaturation and cooling step. After these steps are complete, the meansfor controlling the flow of fluid is operated to allow the reactionsolution to pass through the fluid channel 50 from the first chamber Ato the second chamber B. For example, the valve in the fluid channel isopened and the fluid sample is directed into the second chamber eitherby pressure or vacuum techniques. The reaction solution is then broughtinto contact with the amplification enzyme(s) (e.g. T7 and/or RT) andthe enzymatic amplification process proceeds in the second chamber B at42 degrees C.

In a preferred embodiment, after completion of the amplificationreaction in chamber B, the test device is manually removed from theamplification station 1 of FIG. 1 and inserted into a separatedetection-type instrument. In the detection-type instrument, an SPR® (afluid transfer device which serves as a solid phase receptacle)pipette-like device is introduced into the second chamber. The teststrip 10 contains a plurality of wells arranged in an array.Hybridization, washing, optical analysis and decontamination thenproceeds in the wells 13 in accordance with well known techniques inorder to detect the amplification products. Such processes may occur inthe adjacent wells of a test strip embodiment of the dual chamberreaction vessel automatically in the VIDAS® instrument of bioMerieux,Inc.

Turning now to a detailed description of the construction of the testdevice used in the amplification station, FIG. 1A is a perspective viewof a test device in the form of a strip 10 incorporating a dual chamberreaction vessel 12 for a nucleic acid amplification reaction that meetsthe above requirements. The test strip 10 includes a plurality ofhybridization and wash wells 13, and an associated cover member 14. Thetest strip 10 of FIG. 1 is preferably made from a molded polymericmaterial, such as polypropylene.

A sealing membrane, such as an aluminum film coated with polypropylene,is applied to the upper surface 15 of the test strip to cover the wells13 and dual chamber reaction vessel 12, after the wells and vessel 12have been pre-loaded with the appropriate enzyme, reagent wash or buffersolution, etc. The membrane is not shown in FIG. 1A in order to betterillustrate the structure of the test strip 10. The cover member 14 isshown prior to attachment to the test strip in the vicinity of the dualchamber reaction vessel 12.

The test strip of FIG. 1A can be used in the amplification station ofFIG. 1 to perform an isothermal nucleic acid amplification reaction,e.g., a TMA reaction, in accordance with one possible embodiment of theinvention. Chamber A of the dual chamber reaction vessel 12 contains theamplification reagents or mix, namely deoxynucleotides, primers, MgCl₂and other salts and buffer components in liquid or pellet form. ChamberB is in fluid communication with a enzyme pellet well 52 that containsthe amplification enzyme(s) that catalyzes the amplification reaction,e.g., T7 and/or RT, in liquid or pellet form. In an alternativeembodiment, the amplification enzyme is loaded directly into chamber B.

After addition of the targets (or test sample) into chamber A, the covermember 14 is closed down onto the test strip 10 in the manner to bedescribed and the test strip is installed into one of the bays 3 of theinstrument 1 of FIG. 1. Inside the instrument, heat is applied tochamber A to denature the DNA nucleic acid targets and/or remove RNAsecondary structure. The temperature of chamber A is then quickly cooleddown to allow primer annealing. Subsequently, the solution of chamber Ais brought into contact with the enzyme pellet in the pellet well 52 andthe solution is introduced into chamber B. Chambers A and B, now influid communication with each other, are then maintained at the optimumtemperature for the amplification reaction, e.g., 42 degrees C. Byspatially separating chamber A from chamber B, and applying the heat fordenaturation to chamber A only, the thermolabile enzymes in the enzymepellet well 52 are protected from inactivation during the denaturationstep.

After the nucleic acid amplification reaction is completed, the teststrip 10 is then removed from the instrument 1 of FIG. 1 and processedin a second detection machine adapted to process the test strips, suchas the VIDAS® instrument. The test strip 10 of FIG. 1A is given aparticular form factor (e.g., shape, length, width, height, end features18A and 18B, etc.) so as to enable the test strip to be compatible withan existing instrument base having a solid phase receptacle and otherequipment for processing the results of the nucleic acid amplificationreaction in the test strip per se. Additionally, the form factor of thetest strip will drive the design of the mechanical features in theamplification station 200 of FIG. 1. Thus, while the preferredembodiment of the test strip 10 has a form factor suitable for theinstrument base of the inventors' assignee, it will appreciated that adifferent size, shape, configuration, and other physical characteristicsof the test device incorporating the dual chamber reaction vessel can bearrived at to suit other analytic instruments, and other instrumentsthat would conduct the nucleic acid amplification reaction in the dualchamber reaction vessel. Thus, the inventors do not consider theinvention limited to the particular test strip illustrated in thedrawings.

FIGS. 2 and 3 are additional perspective views of the test strip 10 andcover member 14 of FIG. 1A. FIG. 4 is an isolated perspective view ofthe cover member. Referring to FIGS. 2-4, the cover member 14 has a pairof resilient legs 20 with a wedge feature 21 that snap ontocorresponding ledges 72A formed in the upper edge of the test strip, aswill be explained later in conjunction with FIGS. 7-10. The legs 20allow the rear portion 22 of the cover 14 to be firmly and securelyattached to the test strip 10, while allowing a second or forwardportion 24 of the cover 14 to be raised and lowered relative to the rearportion 22. The cover 14, made of a molded polymeric material, includesan integral hinge portion 26 linking the portions 29 and 24 together.The cover also includes a central aperture 28 having a porous meshfilter placed therein to allow air to enter into or be removed fromchamber A (after removal of the sealing membrane from the top of chamberA), while substantially blocking the escape of fluids or reagents fromchamber A or the entry of foreign matter into chamber A.

The purpose of the cover 14 is to control access by the user to chamberA and to provide a protective barrier from the environment during theperformance of the nucleic acid amplification reaction. Duringmanufacture of the test strip, the reagents are loaded into chambers Aand B (and to the wells 13), and then a sealing membrane is applied tothe surface 15 of the test strip 10, covering all the wells 13 and thechambers A and B. The membrane may be given a perforation or tear lineat a location indicated at 34, adjacent to chamber A. Then, the covermember 14 is installed on the test strip 10. When the technician isready to use the test strip 10, the user lifts up the front portion 24of the cover to the position shown in FIG. 2. The edge 30 has a curvedrecess feature for the user's finger to assist in lifting up portion 24.Then, the technician grasps the free edge 32 of the membrane (shownbroken away in FIG. 2 to illustrate the structure of the test strip),and pulls away the membrane such that the membrane separates at theperforation, indicated at 34. This action exposes chamber A of the dualchamber reaction vessel 12. Then, the technician introduces the fluidsample into chamber A and closes the cover member 14.

Referring to FIGS. 4 a and 4B, optimally and in the preferredembodiment, the film or membrane remains in place over chamber A. Thecover member includes a manually actuated button 41 that has aprojecting point or surface 41B on the underside thereof such that whenthe cover member 14 is closed by the user, the user may actuate anddepress the button 41 and thereby cause the projecting point 41B topierce the membrane covering the top of the test strip above Chamber Ato provide a small opening for the introduction of the test sample. Inthis embodiment, the foil membrane is not removed by the technician butrather is left in place. The action of the button/projecting point isthe mechanism by which chamber A is accessed at the time of use. Thisembodiment reduces the likelihood that any fluid or reaction solutionsmay unintentionally migrate out of the chamber B and into theenvironment. As seen in FIG. 4A, the button 41 is connected to the restof the cover by means of resilient legs 41A which allow the button 41and projecting point 41B to move relative to the cover member andthereby pierce the membrane. Once moved the lower position, the sidewall 41D of the button snugly fits within the corresponding circularwall portion 41E of the cover member 14, shown best in FIG. 4B.

The cover member 14 has an additional pair of resilient gripping legs 38on opposite sides thereof that snap onto rim features 72B on oppositeedges of the test strip, resulting in the secure engagement of the cover14 to the test strip 10. The legs 38 grip the strip 10 with much lessforce than the rear legs 20, thus the cover 14 does not becomecompletely disengaged from the test strip when the user lifts up thefront portion 24 of the cover. A third pair of legs 36 is provided onthe cover and helps align the front portion to the test strip 10 whenthe cover is closed.

Referring to FIGS. 5 and 6, the top surface 15 of the test strip 10includes a aperture 70 designed to accommodate a fork (shown in FIGS.14-16) during the process of opening the connecting conduit 50. Thecover member 14 of FIGS. 3 and 4 is installed over the test strip 10such that the aperture 40 of the cover member is directly over theaperture 70 of the test strip. FIG. 5 also shows the ledge features 72Aand 72B that enable the resilient legs 20 and 38 of the cover member 14to lock onto the test strip when the cover member 14 is installed ontothe test strip. Referring to FIGS. 9 and 10, the test strip has aslanted portion 74 over which the wedge feature 21 (FIG. 4) of the covermember slides until the wedge feature 21 snaps under the ledge 76 andpresses against the wall portion 78. The resilient nature of the legs 20of the cover member and the action of the wedge 21 against the shelf 76prevents the cover member 14 from becoming disengaged from the teststrip during the operation of raising and lowering the front portion 24of the cover member. The slanted surface 80 of FIG. 9 assists ininstalling the cover member and aligning the legs 20 relative to theledge feature 72. The operation of the ledge feature 72B is the same forthe legs 38 of the cover 14.

Referring to FIGS. 6 and 8, the test strip has a pair of transverselyextending ridges 84 molded into the bottom of the test strip that allowthe test strip to be placed in a stable, level attitude on a table top.

FIGS. 2 and 8 illustrate a base cap 86 that is manufactured separately.The cap 86 is ultrasonically welded to the base of the chamber A, to aweb 87 linking the chamber A to the connecting conduit 50, and to thebase of the connecting conduit 50. The cap 86 covers the extremelowermost portion of chamber A and provides a fluid pathway for solutionto pass from the base of chamber A to the base of thevertically-disposed connecting conduit 50. The cap 86 is basically thesame construction as those cars performing a similar function in theU.S. Pat. No. 5,786,182, which is incorporated by reference herein.

As shown best in FIGS. 5, 8 and 11, the test strip 10 further includes apair of desiccant wells 54 and 56 which are placed in air or fluidcommunication with chamber B. The desiccant well 54 is also shown inFIG. 6, which is a cross-sectional view of the test strip taken alongthe lines 6-6 of FIG. 5. The desiccant wells 54 and 56 are designed tohold one or a plurality of small desiccant pellets stacked on top ofeach other in their respective wells. During assembly of the test strip,machine inspection of the desiccant wells will confirm the quantity ofdesiccant pellets in the wells 54 and 56. The purpose of the desiccantis to extend the shelf life of the amplification enzyme loaded into thetest strip, particularly where the amplification enzyme is in a pelletform and susceptible to degradation in the presence of a moistenvironment. In the event that the nucleotides, MgCl₂, primers and otherreagents loaded into chamber A are in liquid form, then the desiccantwells 54 and 56 need not be placed in direct air or fluid communicationwith chamber A. However, in the event that the reagents in chamber A arein pellet form or otherwise susceptible to degradation in a moistenvironment, then the desiccant wells will be designed and constructedto communicate with chamber A in addition to chamber B. Alternatively, asecond set of desiccant wells can be provided adjacent to chamber A toservice the reagents in chamber A.

Referring in particular to FIGS. 6 and 11, the extreme lateral portionof the desiccant well 54 includes a passageway indicated 58 allowing aircommunication with the chamber B (and ultimately air communication withthe enzyme pellet placed in the enzyme pellet well 52). The passageway58 is provided above a wall 60 that separates the lateral portion of thedesiccant well 4 from chamber B. Three or four desiccant balls 62 areplaced in the desiccant well 54. Alternatively, the desiccant ballscould be directly placed in chamber B (and Chamber A as necessary), ormolded into the material forming the dual chamber reaction vessel 12.

After the denaturation and primer annealing of the fluid sample inreaction vessel A has taken place at the first reaction temperature, aball valve, indicated generally by reference numeral 102 in FIGS. 12 and13, is opened. The ball valve consists of a metal ball 104 that isdisposed in the connecting conduit 50 in a cylindrically-shapedintermediate region 106. The ball 104 is sized such that its diameter isequal to the diameter of the intermediate region 106, thus it normallyforms a complete obstruction of the connecting conduit. The walls 108 ofthe connecting conduit 50 are made from a deformable material (andpolypropylene is sufficiently deformable for the present purposes). Thisdeformability of the walls 108 is such that, when the walls 108 aresqueezed on opposite sides of the ball 104, the wall 108 is deformed inthe direction perpendicular to the squeezing force, on opposite sides ofthe ball, to thereby create a passage for fluid around the ball.

A fork is provided in the amplification station 200 to create thisdeforming action on the walls 109 and ball 104. The fork 110 has twoprongs 112 for each position, i.e., six forks with a total of twelveprongs per bay, for a bay designed to contain six test strips at any onetime. The fork 110 is lowered through the aperture 40 of the cover (asbest shown in FIG. 14), through the aperture 70 in the top of the teststrip (shown best in FIG. 11), such that the prongs 112 come intosqueezing contact with the walls 108 of the connecting conduit 50directive on opposite sides of the ball 104, as shown best in FIG. 16.This squeezing action deforms the walls 108, as shown in FIG. 17 to formpassages 116 on opposites sides of the ball 104.

Simultaneous with or immediately after the opening of the ball valve asjust described, a vacuum is drawn on the test strip, and particularly onthe first reaction chamber A. This is achieved by placing a vacuumenclosure around the test strip in the bays 3 of the amplificationstation 1 (described in more detail later on), and evacuating the air inthe vacuum enclosure. The drawing of the vacuum lowers the pressure inboth the first and second chambers A and B, since they are now in airand fluid communication with one another. When the vacuum is released, apressure gradient exists between chamber A and chamber B, with chamber Aat a higher pressure. The pressure gradient forces the fluid solution inchamber A through the passage in the cap 86 (see FIGS. 12 and 13), upand around the passages 116 in the connecting conduit 50 as indicated bythe arrows in FIG. 17, and up to the top of the connecting conduit 50.

Once the fluid solution has reached the top of the connecting conduit50, the fluid enters a channel 100 (see FIGS. 11 and 12) leading to theenzyme pellet well 52. The fluid dissolves the enzyme pellet 130 (FIG.12) in the well 52, and carries the amplification enzyme into chamber B.The amplification of the nucleic acid in the fluid sample occurs inchamber B at the specified temperature, e.g., 42 degrees C.

Referring now to FIGS. 14 and 15, the reciprocating action of the fork110 opening the ball valve in shown schematically. In FIG. 14, the teststrip 10 is shown with the sealing membrane 42 applied to the topsurface of the test strip in the manner described previously, as itwould be when the device is manufactured and ready for use. The membrane42 carries a bar code 43 identifying the type of test strip that isbeing used or other pertinent information.

In FIG. 15, the basic features of operation of the forks 110 in theamplification station 1 of FIG. 1 is shown. In FIG. 15, two test strips10 are shown installed in the amplification station, shown in an endview an partially in section. The forks 110 are shown as being integralwith a cross-member 132 that is in turn bolted to the top of a vacuumcover housing 134 in the amplification station 1. The test strips 10 areinstalled on a TEC/heat sink assembly 136 that maintains the twochambers of the dual chamber reaction vessel in the test strips 10 atthe proper temperature, as described in detail in the U.S. Pat. No.5,786,182. The vacuum cover housing 134 is attached to a mechanicaldrive mechanism that raises and lowers the vacuum cover housing relativeto a lower support structure 138. The cover housing 134 and supportstructure 138 define a vacuum enclosure or chamber 140. The vacuum coverhousing 134 further includes ports (not shown) for withdrawing air fromthe vacuum enclosure 140 and introducing air back into the vacuumenclosure 140. When the vacuum cover housing 134 is lowered down ontothe support structure 138, it forms an air-tight seal with the supportstructure 138 (using a suitable gasket structure in the region 139),enabling vacuum to be drawn in the enclosure. The drawing of vacuum inthe enclosure 140 causes air to be withdrawn from the dual chamberreaction vessel via the aperture 28 in the cover member 14 and anair-permeable filter 142 placed therein (see FIG. 14). Then, when thevacuum is released in the enclosure 140 (the housing 134 remaining inthe lower position during the release of vacuum) the pressuredifferential between chambers A and B causes fluid solution in chamber Ato migrate through the connecting conduit, opened by the action of theforks 110, and into the enzyme pellet chamber and chamber B, in themanner described previously.

Further details on the presently preferred lest strip 10 are set forthin the patent application of Bryan Kluttz et al. filed concurrently,Ser. No. 09/420/139 now U.S. Pat. No. 6,410,275 entitled “DisposableTest Devices for Performing Nucleic Acid Amplification Reactions”,incorporated by reference herein.

II. Detailed Discussion of Amplification Station

Overview

Referring now to FIGS. 18 and 19, the top cover of the amplificationmodule 2 is shown removed in order to better illustrate the twoidentical amplification stations 200 placed immediately behind the bays3. The amplification module 2 also includes a pair of glass jars 202 andassociated components of a pneumatic system 204 for the stations 200,described subsequently in conjunction with FIG. 46.

One of the amplification stations 200 of FIGS. 18-19 is shown in aperspective view in FIG. 20. FIGS. 20-27 are a set of elevational, planand perspective views of the amplification station 200. Referring tothese figures, together with FIGS. 1 and 2, the amplification stationincludes a support structure 206 that is adapted to receive one to sixof the disposable test devices 10 of FIGS. 1A-17. In particular, thesupport structure 206 includes a set of raised ridge elements 208 thateach have a groove 210 (FIG. 21). The grooves 210 extending the lengthof the ridges 208 and receive the outwardly-projecting cylindricalfeatures in the end 18B of the test strips 10 of FIG. 2. The test stripsare manually inserted the bay 3, with end 18B inserted first, such thatthe strips are held in place by the action of the ends 18B being held bythe grooves 210 in the raised ridge elements 208.

The amplification station 200 includes a temperature control system forthe test strips. The temperature control system is described inconjunction with FIGS. 34, 35, 37 and 38. Basically, the temperaturecontrol system consists of thermoelectric heating elements, associatedheat sinks, and a feed-back control system. The temperature controlsystem maintains chamber A of the test strip at a first elevatedtemperature for purposes of denaturation of the sample in chamber A ofthe test strip 10. The temperature control system simultaneouslymaintains the amplification enzyme in the enzyme pellet well at a secondtemperature lower than the first temperature, so as to preserve thesecond nucleic acid amplification reagent (i.e., prevent inactivation ofthe amplification enzyme). The temperature control system also maintainschamber B of the test strip at the desired temperature for theamplification reaction performed therein. The thermo-electric heatingelements are placed in thermal and physical contact with the supportstructure 206 immediately adjacent to the test strips, and transfer heatto or remove heat from the reaction chambers of the test strips.

The amplification station 200 also includes an actuator that isoperative on the test strip 10 to place the first and second reactionchambers in fluid communication with each other. The actuator isoperative on the test strip after a reaction has occurred in the firstreaction chamber A at the first elevated temperature. The constructionof the actuator will vary depending on the design of the test device. Inthe preferred test strip embodiment, the actuator consists of a fork 110having two prongs or tines 112. In the instant embodiment, there are sixsuch forks 110 (one per test strip). The forks are best shown in FIGS.24 and 2S. The forks are mounted to upper surface of a vacuum housing134, and reciprocate up and down with the vacuum housing 134 relative tothe support structure 206 and test strips in the manner described ingreater detail below.

In a preferred embodiment, the amplification station includes apneumatic system that promotes the transfer of a reaction solution fromthe first chamber A of the test strip to the second chamber B. Onepossible implementation of the pneumatic system is to use vacuum probesthat draw a vacuum on the second chamber B of the test strip. The vacuumdraws fluid from chamber A through the connecting conduit 50 in the teststrip into the second chamber B. This technique is described at lengthin U.S. Pat. No. 5,786,182 which is incorporated by reference herein.

In a more preferred embodiment, the entirety of the test strip, andindeed the entirety of all six of the test strips, are placed in avacuum enclosure and vacuum is drawn on the test strip. The release ofvacuum causes the fluid to be transferred from chamber A to chamber Bdue to a pressure differential between the two chambers. Theamplification station 200 includes a pneumatic system (illustrated inFIG. 47 and described later on) that generates and releases a vacuum ina vacuum enclosure defined by the upper vacuum chamber housing 134 (seeFIGS. 23-26) and the support structure 206. The upper vacuum chamberhousing 134 moves up and down by a drive system between a raisedposition, shown in FIG. 23, and a lower position. In the lower position,a gasket 220 (FIG. 29C) held in the gasket retaining feature 222 of thevacuum chamber housing 134 seats on the planar peripheral surface 224 ofthe support structure 206. The gasket 220 forms an air-tight sealbetween the vacuum chamber housing 134 and the support structure,allowing a vacuum to be drawn inside the vacuum chamber housing. Whenthe vacuum chamber housing 134 is lowered onto the support structuresurface 224, the forks 110 operate to open the valves of the test stripin the manner indicated in FIG. 1A. As is evident from FIGS. 20-27, allof the test strips loaded into the amplification station aresimultaneously subject to valve actuation and pneumatic transfer offluid from reaction chamber A to the reaction chamber B in the teststrips.

It is important that the support structure 206 and in particular theperipheral surface 224 thereof be absolutely level, so that when thevacuum housing 134 is lowered onto the support structure 206 a tightseal is formed by the gasket 220. It has been found that, by loosening acollar 226 at the top of the guide screws for the vacuum housing drivesystem, the vacuum housing 134 has enough play to uniformly settle onthe support structure and form a vacuum seal.

Additional Mechanical Features of Amplification Station 200

Referring in particular to FIGS. 20, 22 and 23, the amplificationstation 200 includes a door 230 that is mechanically fastened to thevacuum housing 134 and reciprocates up and down therewith. When the dooris in the raised position shown in the drawings, the user is able toinsert the test strips into the bay 3 of FIG. 1 and into the ridges 208of the support structure 206. A sensor plate 232 is also mechanicallyfastened to the vacuum housing. The sensor plate 232 has a flange 234that moves up and down within an opening 236 in a structural supportmember 237. Three optical interrupt sensors 238A, 238B and 238C aremounted to a side panel 240 of the station and detect the passage ofupper and lower edges 242 and 244, respectively, of the sensor plate232. The optical interrupt sensors 238A-C supply signals to digitalelectronic control system for the station and are used to monitor andcontrol the raising and lowering of the door 230 and vacuum housing 134.

The top of the amplification station includes a tray 250 having anoptional air filter 252. The air filter 252 filters air in the air inletline 254 leading to the vacuum housing 134. The tray 250 also carriestwo solenoid valves 256A and 256B that control the drawing and releaseof vacuum in lines 254 and 258 leading to the vacuum housing 134. Theoperation of the valves 256A and 256B will be discussed later. A line260 leads from the vacuum chamber housing port 292 to a pressure sensormonitoring the pressure inside the vacuum chamber housing.

Referring now to FIGS. 21, 24-26 and 31, an optical reader assembly 270is mounted above the rear of the support structure 206. The opticalreader assembly 270 includes up to six optical sensors per position thatare positioned directly over the spaces 272 between the ridges 208 inthe support structure 206. The optical sensors detect whether the userhas inserted a test strip into the support structure 206, as such teststrips will occupy the spaces 272. The optical reader assembly is shownisolated in several views in FIGS. 32A-32D.

Referring to these figures and primarily to FIG. 31, the optical readerassembly 270 includes a cable 274 for the optical sensors. The cable 274has a plug 278 that connects to another cable leading to the electroniccontrol system for the station. The cable 274 leads to a housing 276that is received in an aperture in the support structure 206. Thehousing 276 is retained against the support structure 206 by a C-clip280. A gasket 282 prevents air from leaking around the side of thehousing 276 during the vacuum operations. The cable 274 leads to sixoptical sensor arrant 284 located inside a cover 286.

Referring to FIGS. 26, 27 and 31, the vacuum housing 134 includes aprojecting portion 290 that receives the housing 276 when the vacuumhousing is lowered onto the support structure 206. The vacuum housing134 is shown isolated in FIGS. 29A and 29B. The vacuum housing 134includes three ports 292, 294 and 296. Port 292 receives a tube 250(FIG. 20) that leads to a pressure sensor monitoring the air pressureinside the vacuum housing 134 when the housing 134 is lowered onto thesupport structure 206. Port 294 receives the tube 258 that leads to thesolenoid valve 256B of FIG. 20. Air is drawn out of the vacuum enclosureprovided by the vacuum housing 134 via the port 294 and its associatedtube 256. Port 296 receives a third tube 254 that leads to the solenoidvalve 256A of FIG. 20. Air is reintroduced into the vacuum enclosure viathe port 296.

The vacuum housing 134 also receives a negative temperature coefficienttemperature sensor 300. In this type of sensor, when the sensedtemperature increases, the resistance value decreases. The temperaturesensor 300 has leads 302 conducting voltage signals to the electronicsand temperature feedback control system for the station described inmore detail below. Basically, the feedback provided by the ambienttemperature sensor 300 allows for compensation for a drift intemperature of the support structure due to heating of the ambient airin the vacuum chamber.

Referring to FIGS. 20, 23, 28, 29A and 29C, the vacuum housing 134 alsoincludes apertures 304 for receiving a pair of bolts 306. The bolts 306secure the vacuum housing 134, the cross-member 132, and the forks 110to a horizontally-oriented support member 308. A pair of O-rings 309prevent air from entering around the cross-member 132 in the vicinity ofthe bolts 306. The support member 308 is fastened at opposite sidesthereof to a guide collar 310 that is raised and lowered by therotational action of a lead screw 312 driven by a motor and belt drivesystem indicated generally at 314. See also FIG. 40.

Referring now to FIGS. 20, 21 and 23, a pair of fans 320 are provided inthe lower portion of the station. The fans 320 direct air to the spacebelow the horizontal support member 206, and in particular over a set offins 322 providing a heat sink for the thermoelectric elements in thetemperature control system for the station.

Referring now to FIGS. 36, 33A and 33B, the entire support structure 206including attached heat sink fins 322, is shown isolated in perspectiveviews. FIG. 33C is a top plan view of the support structure 206. Thesupport structure 206 includes a tray support 207. The tray support 207includes three guide collars 324, two on one side and one on the other.The guide collars 324 receive a shaft extending from the front of thestation to the rear of the station. The shafts are shown in FIGS. 24 and25 as reference 326. As shown in FIG. 36, the guides 324 include aplastic, low friction insert 328. A coil spring 330, shown best in FIGS.20, 22, 23, is provided between the end of the guide collar and thesuperstructure of the station. The coil springs 330, guide collars 324,and shafts 326 allow the entire support structure to move back and forthalong the axis of the shafts 326 for purposes of agitation and mixing ofreaction solution in the test strips to completely dissolve the enzymepellet. The back and forth action of the support structure 206 forpurposes of agitation and mixing is provided by a motor, belt, andeccentric gear assembly, described in further detail below.

As shown in FIGS. 33B and 33C, the support structure includes a pair ofupright flanges 340. The cover 286 of the optical read assembly 270 ofFIG. 32A is mechanically fastened to the flanges 340. Thus, the sensorsof the optical read assemble are positioned directly above the spaces272 between the raised ridges 208. FIG. 33C also illustrates the sixpairs of recessed regions 342 in the front portion of the supportstructure 206. The recessed regions 342 are designed to allow the prongs112 of the forks 110 (FIG. 28) to be fully inserted into the teststrips, without bottoming on the base of the support structure 206 anddamaging the forks. FIG. 33C also shows an aperture 344 in the supportstructure that receives the housing 276 of the optical read assembly(see FIG. 31).

Referring now to FIGS. 28 and 30A-30D, the cross-member 132 and forks110 of FIG. 28 are shown isolated. The cross-member 132 has a pair ofrecesses 354 for receiving an O-ring 309 (FIG. 29) forming a seal forthe vacuum housing. A cylindrical raised feature 356 receives the bolts306 of FIG. 28 that fasten the cross-member to the primary horizontalspan member. The cross-member 132 and integral forks 112 and prongs 112is made from high grade stainless steel in order to withstand the forcesrequired to open six of the ball valves in six test strips, over thelife of the instrument.

The cross-member 132 further includes a set of six spring-loadedpositioning prongs 360. The positioning prongs 360 are moveable within acylindrical recess 362 in the cross-member 132 against the force of abiasing spring 364. The positioning prongs 360 press down on the cover14 of the test strips 10 (FIG. 2) to help the cover 14 form a sealaround chamber A in the test strip. The purpose is so that when air isevacuated from the chambers A and B of the test strip during the vacuumprocedure, and then reintroduced into the chambers when the vacuum isreleased, the air passes through the porous mesh filter 142 (FIG. 14) inthe cover 14 and not around the edges of the cover member. The springs364 limit the amount of force applied to the cover 14 to about 3 poundswhen the fork and vacuum chamber 134 is lowered onto the test strips andsupport structure 206, preventing the cover from breaking.

Referring now to FIGS. 24 and 25, the station 200 sits upright insidethe amplification module 2 of FIG. 1 by means of two legs 352 and a footpad 354.

Temperature Control System Operational Features

Referring now to FIGS. 33B, 34 and 35, the general operation of thetemperature control system for the station will be described. FIG. 34 isa bottom plan view of the station 202, with all of the drive motors andother components removed in order to more clearly show the basicfeatures of the temperature control system. The support structure 206can be conceptually divided into two temperature-controlled regions, afirst region 370 and a second region 372. The region 370 is devoted toheating chamber A of the test strip to a first, elevated temperature,e.g., ≧65 degrees C., for denaturation of the sample. The region 372 isdevoted to heating chamber B of the test strip to a second temperature,lower than the first temperature, in order to preserve the integrity ofthe amplification enzyme in the enzyme pellet well and conduct anamplification reaction in chamber B of the test strip at the desiredtemperature, e.g., approximately 42 degrees C.

The region 370 is maintained at the first temperature by virtue of twothermo-electric cooler (TEC) elements 374A and 374B that are in physicaland thermal contact with the front portion of the ridges supporting thetest strips. Thermo-electric coolers 374A and 374B are in physical andthermal contact with the heat sink fins 322. The thermo-electric coolers374A and 374B are positioned between the fins 327 and the top surface ofthe support stricture, as will be described later in conjunction withFIGS. 37 and 38. Thermally sensitive resistors i.e., thermostatsembedded in the support structure and the heat sinks provide feedbackinto the computer control system.

Likewise, the temperature of the region 372 is controlled by twothermo-electric coolers 376A and 376B, physically and thermally incontact with the rear portion of the ridges supporting the test stripsand with the cooling fins or heat sink 322.

FIG. 35 illustrates schematically the operation of the thermo-electriccoolers. Basically, a thermoelectric cooler is a solid state device thatfunctions as a heat pump without any moving parts, fluids or gasses.Thermoelectric coolers are made up of two semiconductor elements,primarily Bizmuth Telluride, heavily doped to create either an excess(N-type) or deficiency (P-type) of electrons. The heat absorbed at thecold junction is pumped to the hot junction at a rate proportional tothe current passing through the circuit and the number of couples. Atthe cold junction, the electrons absorb the energy (heat) as they passfrom a low energy level in the P-type semiconductor element, to a higherenergy level in the N-type semiconductor element. The DC power supplyprovides the energy to move the electrons through the system. At the hotjunction, the energy is expelled to a heat sink as electrons move fromthe high energy level element (N-type) to a lower energy level element(P-type). By inversion of the DC source polarity, the heat sink becomesthe heat source and the heat source becomes the heat sink. Thus, thethermoelectric coolers of FIGS. 34 and 35 can be used to both heat andcool the support structure and the test strips in accordance with adesired temperature profile for a nucleic acid amplification reaction.The thermo-electric cooling elements 374A, 374B, 376A and 376B of FIG.34 are available commercially.

Referring to FIG. 37, the support structure 206 and temperature controlsystem is shown in a cross-sectional view taken along the lines 37-37 ofFIG. 34. FIG. 37 shows two TEC modules 374A and 376A, positionedimmediately above and in thermal contact with the fins (heat sinks) 322.The front TEC module 374A is responsible for bringing the front portionof the support structure 206 in region 370 to a first higher temperaturetypically greater than 65 degrees C. as described above. The TEC module376A likewise is in thermal and physical contact with the rear set ofheat sink fins 322 and maintains the region 372 of the support structureat a second temperature e.g. 42 degrees C.

FIG. 38 is a more detailed cross-sectional view of the regions 370 and372 of FIG. 37. A thermistor 400 is embedded into the heat sink 322 andmonitors the temperature of the heat sink for the temperature controlfeedback system. The TEC module 374A is sandwiched between the heat sink322, an electrical insulator 401, and a plastic tray 402 forming thefront portion of the support structure 206. A bolt 404 secures theassembly 322, 374A, 402 and 206. A gasket 406 prevents air or fluid fromleaking around the plastic tray 402. A second thermistor 408 embedded inthe front region 410 of the raised ridge 208 monitors the temperature ofthe support structure in the region immediately adjacent to the chamberA of the test strip. The second thermistor 408 is mounted inside theraised ridge 208 by means of a plastic platform 412 extending across thesupport structure and secured in place by a fastener assembly 414.

The platform 412 and a second fastener assembly 416 also secure a thirdthermistor 418. The two thermal regions 370 and 372 of the supportstructure raised ridge 208 are separated from each other by means of aninsulative Delrin spacer 420, air gaps 422, and locating screw 424.

Referring to the left hand side of FIG. 33, the rear thermal region 372includes the TEC 376A, and electrical insulator 426, an O-ring gasket428 and a fastener 430 securing the assembly together.

Referring again to FIG. 37, it will be seen that the raised ridge 208 ofthe support structure 206 includes an thermally conductive aluminumblock 432 for the rear or “amplification” thermal region 372 (forchamber B of the test strip and the amplification enzyme), and a secondthermally conductive aluminum block 434 for the front or “sample”thermal region 370 (for chamber A). The material chosen for the rearmostportion 436 of the raised ridge is not particularly important, as itdoes not perform any heat transfer functions in the illustratedembodiment.

FIG. 37 also shows a circuit board 433 containing the electronics forthe two sample fans 320 of FIG. 34 and the TEC modules 376A-B and374A-B.

Referring to FIG. 39, the support structure 206 and associated thermalcontrol system components are shown in another cross-sectional view,taken along the lines 3939 of FIGS. 33C and 34. The entire sample heatsink 322 is mounted to the sample thermal block 434 by means of bolts440 and 404. A tension spring 442 and a gasket 444 are provided atopposite sides of the assembly to limit the amount of force applied tothe TEC modules 374A and 374B by the bolts 440 and 404.

Agitation and Belt Drive System Operational Features

FIG. 40 is a perspective view of the superstructure of the station 200with most of the parts thereof removed in order to better illustrate thedrive systems of the station. The drive systems consist of two separateassemblies: (1) a belt drive system 500 for raising and lowering thevacuum chamber housing relative to the support structure, and (2) anagitation drive system 502 for causing back and forth movement of thesupport structure along the axis of the shafts 326 (see FIG. 22).

Referring to FIGS. 40, 28, 42 and 43, the belt drive system 500 includesa stepper motor 504 driving a toothed belt 506 that rotates a pair ofgears 508 and attached lead screws 312. Rotation of the lead screws 312within the collar 310 causes the horizontal support member 308, collar310 and attached vacuum chamber housing 134/fork 110 assembly to move upand down relative to the lead screws. The optical sensors 238A-C of FIG.22 sense the position of the drive system 500 by monitoring whether thesensor panel 234 is obstructing the path of light across the sensor.

Referring to FIGS. 26, 40 and 42-44, the agitation drive system 502includes a stepper motor 550, toothed belt 554 and an eccentric gearassembly 556. An optical sensor 558 detects the position of a cut-out560 in a disk 562 attached to the gear 556 and generates a signal usedby the motor 550 to return the eccentric gear 556 to a home position.The eccentric gear abuts a block 564 mounted to the underside of thesupport structure 206 (shown best in FIGS. 28, 33B and 37) and is heldagainst the block 564 by the action of the coil springs 330 (FIGS. 23,25) surrounding the shafts 326. Rotation of the eccentric gear 556causes a back and forth movement of the entire support structure 206,causing a shaking motion to be imparted to the test strips loaded on thesupport structure, facilitating complete dissolution of the pellet andpromoting a mixing or the reagents with the fluid sample in the teststrips.

The motion of the agitation system is approximately 8-10 hertz with a 3mm stroke +/−1.5 mm. The agitation occurs for 60 seconds, and startswhen the forks and vacuum chamber housing are raised by the drive system500, after fluid has transferred from chamber A to chamber B in the teststrips. The agitation thus promotes the reaction between the reactionsolution coming from chamber A with the amplification enzyme.

As shown in FIGS. 43 and 44, the eccentric gear 556 extends through anaperture in a base or platform 570 for the station. The base 570includes a pair of upraised guides 572 for supporting the shafts 326 ofFIG. 22.

Electronics System Operational Features

Referring to FIG. 45, the electronics system 600 for the amplificationstation 200 is shown in block-diagram form. The electronics system 600includes a front tray board 602 that receives signals from passivetemperature sensors in the front part of the support structurecorresponding to temperature region 370, and supplies the signals to atray interface board 604. A rear tray board 606 receives signals fromthe passive temperature sensors in the rear portion of the supportstructure corresponding to temperature region 372 and supplies them tothe tray interface board 604.

A servo board 610 controls the active components of the station,including the vacuum valves in the pneumatic system, the fans, and themotors for the drive systems. The servo board 610 also issues commandsto the optical sensors in the optical reading system to detect whether astrip has been loaded into any given slot of the support structure.There is one servo board 610 per bay.

An interface board 612 is responsible for a variety of tasks, includingcontrol of the servo board via RS 485 communication, communication withthe external general purpose computer system 5 of FIG. 1, vacuum supply,and management of Ready and Power On LED's. The interface board 612includes a 68HC11a microcontroller, a flash memory storing software fromthe computer system every time the station is switched on, a RAM storingprogram data, a driver/receiver providing an interface between themicrocontroller and the servo boards 610, another driver/receiverproviding an interface between the microcontroller and the computersystem 5, a voltage reference, providing a measurement of the vacuuminside the vacuum tanks of the pneumatic system, MOS transistorsproviding power supplies for the vacuum motor pump and atmospherevalves, and a fuse providing 12 Volt protection.

The details of the electronics system are not considered pertinent tothe present invention and can be readily developed by persons skilled inthe art.

The servo board 610 controls the whole temperature cycle process orderedby the interface board 612. The four temperature sensors in theinstrument (vacuum chamber ambient temperature sensor, heat sinktemperature sensor, and front and rear temperatures sensors in thesupport structure) provide the measurements to control the temperatureprocess. All of these sensors are negative temperature coefficient (NTC)thermistors, as explained above. Temperature acquisition is by amicrocontroller on the servo board polling a 12 bit A/D converter forthe value of any of the temperature sensors. The voltage valuerepresents sensor impedance, which can be correlated to a temperaturereading.

The temperature control system further includes four power MOSFETtransistors which provide each TEC module with positive or negativevoltage. The microcontroller on the servo board 610 manages a driverthat controls the eight total power MOSFET transistors. Each TEC iscontrolled independently.

Pneumatic System Operational Features

The pneumatic system 204 of FIGS. 18 and 19 is shown in schematic formin FIG. 46. The system 204 serves both bays in the instrument 1. Thesystem includes the vacuum housing 134 forming an enclosure around thetest strips 10 and the support structure 206, a vacuum circuit 700indicated in solid line in FIG. 46 and an atmospheric pressure circuit702 indicated in dashed lines.

The vacuum circuit 700 includes a vacuum pump 704 that holds a vacuum(50 kPA) inside two vacuum tanks 202. A vacuum sensor 706 measures thepressure inside the vacuum tanks 202. The circuit further includes anatmosphere valve EV3. The vacuum tanks 202 are linked to the vacuumhousings 134 for the two bays via a flow reducer 708, a T junction 710,and vacuum lines leading to the vacuum valve EV1 (item 256B in FIG. 20)and vacuum tube 258. Each vacuum housing 134 has a tube 712 leading to avacuum pressure sensor 714 monitoring vacuum inside the vacuum housing134 when it is lowered onto the support structure 206.

The vacuum circuit 700 operates as follows. When the electrovalve EV2 isclosed the vacuum housing is at atmospheric pressure. When the valve EV1is open the air in the vacuum housing flows to the vacuum tanks 202through the flow reducer 708. The flow reducer 708 ensures a gradualdecreasing of the pressure inside the vacuum housing 134.

The atmospheric pressure circuit 702 includes an atmosphere valve EV2(item 256A in FIG. 20) for each bay, a tube 254 leading from the vacuumhousing 134 to a filter 252 and the valve EV2, and a flow reducer 716.

The atmospheric pressure circuit 702 works as follows. The electrovalveEV1 is closed and the vacuum in the vacuum housing is 50 kPa. When theelectrovalve EV2 is open, the ambient air flows to the vacuum housingthrough the flow reducer 716 and the filter 252. The flow reducer 716ensures a gradual increasing of the pressure inside the vacuum housing134.

During initialization of the station 200, the software for theinstrument opens the atmosphere valve EV3 to record the vacuum sensor706 and 714 offset at current atmospheric pressure.

Thermal Cycle

The chamber A of the test strips are heated or cooled by two TEC modules274A and 274B described previously. The same heat sink allows thedissipation of heat from the TEC modules. Similarly, the amplificationreaction chamber B of the test strips is heated and cooled by two TECmodules 276A and 276B, and the heat sink and fins coupled to the TECmodules 276A and 276B allows for the dissipation of heat from theseTECs.

The thermal cycle process carried out by the amplification station 200for a representative embodiment of a nucleic acid amplification reactionfor an amplified Chalmydia trachomatis test is shown in FIG. 47. At timet₀, the temperature of the front portion of the support structure israised to a denaturing and primer annealing temperature of approximately95 degrees C., and maintained there for about 10 minutes. At time t₁,the temperature is rapidly reduced from 95 degrees C. to 42 degrees C.At time t₂, the transfer of reaction solution from chamber A to chamberB occurs in the test strips (the vacuum chamber is lowered onto the teststrips and the vacuum process described above occurs). From time t₂, tot₃ (about sixty minutes), an amplification reaction occurs in chamber Bof the test strips. At time t₃, the temperature in chamber B is quicklyraised to an inactivation temperature of 65 degrees C. at time t₄ andheld there for 10 minutes until time t₅. At time t₅, the temperature isreduced to an idle temperature of 37 degrees C. until the process isrepeated. The test strips are then removed from the bays 3 and insertedinto another instrument for processing of the amplification productswith a probe, solid phase receptacle, or other equipment.

Alternative Implementations

As noted on several occasions above, persons skilled in the art willappreciate that many variations may be made to the preferred andalternative embodiments described above without departure from the truespirit and scope of the invention.

One possible alternative embodiment is to couple the support structurein the bars to an additional drive system that moves the supportstructure relative to the bay door between a retracted position andextended position. The drive system could be of any suitable design. Thesupport structure, in the extended position, protrudes into the dooropening or even further outwardly, thereby enabling a user to moreeasily access the support structure and install the test devices on thesupport structure. When the user has loaded the test devices, they wouldindicate on the user interface that the support structure has beenloaded, whereupon the support structure is withdrawn by the drive systeminto the bay in the position shown in FIG. 20 et seq.

As another example, for certain reactions the amplification station mayonly required to maintain one temperature region in a test device,namely maintain the second reaction chamber at a reaction temperaturesuch as 42 degrees C. Thus, instead of two TEC units and associated heatsinks, only one TEC unit and associated heat sink is provided in theamplification station adjacent to chamber B of the test strips.

This true spirit and scope is to be determined by reference to theappended claims, interpreted in light of the foregoing.

1. A nucleic acid reaction processing station for processing nucleicacid reactions occurring in a plurality of disposable test deviceshaving a nucleic acid sample contained therein and comprising a firstreaction chamber and a second reaction chamber and a valve connectingthe first reaction chamber to the second reaction chamber, said stationcomprising a) a plurality of individual disposable test device receivinglocations, each of said locations adapted for receiving an individualone of said disposable test devices; b) a plurality of actuators, oneactuator per disposable test device, operative on the valve of saiddisposable test devices to open fluid communication between said firstreaction chamber and said second reaction chamber; c) a pneumatic systemincluding a source of vacuum connected to a vacuum enclosure moveableinto a position to cover the disposable test devices and form a vacuumchamber for said disposable test devices, wherein the plurality ofactuators are positioned within the vacuum enclosure, the pneumaticsystem operative to transfer a reaction solution contained in the firstreaction chamber to the second reaction chamber after opening of thefluid communication between the first reaction chamber and the secondreaction chamber; d) at least one thermal module comprising 1) athermo-electric heat source, 2) a first thermal transfer structurepositioned adjacent to at least one of said plurality of individualdisposable test device receiving locations so as to be in direct thermalcontact with said first reaction chamber of a disposable test devicereceived in said disposable test device receiving location, and 3) asecond thermal transfer structure positioned adjacent to at least one ofsaid plurality of individual disposable test device receiving locationsso as to be in direct thermal contact with said second reaction chamberof a disposable test device received in said disposable test devicereceiving location, e) a temperature control system operative of saidthermo-electric heat source so as to produce a desired temperatureprofile to said first thermal transfer structure and thereby a firstdesired temperature profile to said first reaction chamber and a seconddesired temperature profile to said second thermal transfer structureand thereby a second desired temperature profile to said second reactionchamber, in accordance with a nucleic acid reaction to be performed on asample contained in the disposable test device; and f) an optical readerassembly comprising an optical sensor positioned to detect a disposabletest device received within an individual disposable test devicereceiving location.
 2. The amplification station of claim 1, whereinsaid disposable test device further comprises an enzyme pellet well influid communication with said second reaction chamber.
 3. The station ofclaim 2, wherein the pneumatic system is operative to draw a reactionsolution from said first reaction chamber through said enzyme pelletwell to said second reaction chamber after said actuator has operated onthe valve of said disposable test device to thereby deliver an enzymecontained in the enzyme pellet well into said second reaction chamber.4. The station of claim 1, wherein said pneumatic system furthercomprises a drive mechanism for reciprocating said vacuum enclosurebetween upper and lower positions relative to said plurality ofindividual disposable test device receiving locations.
 5. The station ofclaim 1, wherein said station comprises at least four separateindividual disposable test device receiving locations, each of which isin direct thermal contact with said first and second thermal transferstructures.
 6. The station of claim 5, wherein said station comprises atleast four separate optical sensors positioned to detect a disposabletest device received within each individual disposable test devicereceiving location.
 7. A nucleic acid reaction processing station forprocessing nucleic acid reactions occurring in a plurality of disposabletest devices having a nucleic acid sample contained therein andcomprising a first reaction chamber, a second reaction chamber, chambercontaining an enzyme, and a valve connecting the first reaction chamberto the chamber containing the enzyme and the second reaction chamber,said station comprising a) a plurality of individual disposable testdevice receiving locations, each of said locations adapted for receivingan individual one of said disposable devices; b) a plurality ofactuators, one per test device, operative on the valve of saiddisposable test devices to open fluid communication between said firstreaction chamber, said second reaction chamber and said chambercontaining an enzyme; c) a pneumatic system including a source of vacuumconnected to a vacuum enclosure moveable into a position to cover thedisposable test devices and form a vacuum chamber for said disposabletest devices, wherein the plurality of actuators are positioned withinthe vacuum enclosure, the pneumatic system operative to transfer areaction solution contained in the first reaction chamber to the chambercontaining the enzyme and the second reaction chamber after actuation ofthe valve; d) at least one thermal module comprising 1) athermo-electric heat source, and 2) at least one thermal transferstructure positioned adjacent to at least one of said plurality ofindividual disposable test device receiving locations so as to be indirect thermal contact with said second reaction chamber of a disposabletest device received in said disposable test device receiving location,e) a temperature control system operative of said thermo-electric heatsource so as to produce a desired temperature profile to said at leastone thermal transfer structure and thereby a desired temperature profileto said second reaction chamber of said disposable test device inaccordance with a nucleic acid reaction to be performed on a samplecontained in the disposable test device; and f) an optical readerassembly comprising an optical sensor positioned to detect a disposabletest device received within an individual disposable test devicereceiving location.
 8. The station of claim 7, wherein the pneumaticsystem is operative to draw a reaction solution from said chambercontaining an enzyme to said second reaction chamber after said actuatorhas operated on said valve to place said first and second reactionchambers and said chamber containing an enzyme in fluid communicationwith each other to thereby deliver an enzyme contained in the enzymechamber into said second reaction chamber.
 9. The station of claim 7,wherein said pneumatic system further comprises a drive mechanism forreciprocating said vacuum enclosure between upper and lower positionsrelative to said plurality of individual disposable test devicereceiving locations.
 10. The station of claim 7, wherein said stationcomprises at least four separate individual disposable test devicereceiving locations, each of which. is in direct thermal contact withsaid at least one thermal transfer structure.
 11. The station of claim10, wherein said station comprises at least four separate opticalsensors positioned to detect a disposable test device received withineach individual disposable test device receiving location.